The present invention is related to mass spectrometry methods for detecting binding interactions of ligands to substrates and in particular to methods for determining the mode of binding interaction of ligands to substrates.
Drug discovery has evolved from the random screening of natural products into a combinatorial approach of designing large numbers of synthetic molecules as potential bioactive agents (ligands, agonists, antagonists, and inhibitors). Traditionally, drug discovery and optimization have involved the expensive and time-consuming process of synthesis and evaluation of single compounds bearing incremental structural changes. For natural products, the individual components of extracts had to be painstakingly separated into pure constituent compounds prior to biological evaluation. Further, all compounds had to be analyzed and characterized prior to in vitro screening. These screens typically included the evaluation of candidate compounds for binding affinity to their target, competition for the ligand binding site, or efficacy at the target as determined via inhibition, cell proliferation, activation or antagonism end points. Considering all these facets of drug design and screening that slow the process of drug discovery, a number of approaches to alleviate or remedy these matters, have been implemented by those involved in discovery efforts.
The development and use of combinatorial chemistry has radically changed the way diverse chemical compounds are synthesized as potential drug candidates. The high-throughput screening of hundreds of thousands of small molecules against a biological target has become the norm in many pharmaceutical companies. The screening of a combinatorial library of compounds requires the subsequent identification of the active component, which can be difficult and time consuming. In addition, compounds are usually tested as mixtures to efficiently screen large numbers of molecules.
A shortcoming of existing assays relates to the problem of xe2x80x9cfalse positives.xe2x80x9d In a typical functional assay, a false positive is a compound that triggers the assay but which compound is not effective in eliciting the desired physiological response. In a typical physical assay, a false positive is a compound that attaches itself to the target but in a non-specific manner (e.g. non-specific binding). False positives are particularly prevalent and problematic when screening higher concentrations of putative ligands because many compounds have non-specific affects at those concentrations. Methods for directly identifying compounds that bind to macromolecules in the presence of those that do not bind to the target could significantly reduce the number of xe2x80x9cfalse positivesxe2x80x9d and eliminate the need for deconvoluting active mixtures.
In a similar fashion, existing assays are also plagued by the problem of xe2x80x9cfalse negatives,xe2x80x9d which result when a compound gives a negative response in the assay but the compound is actually a ligand for the target. False negatives typically occur in assays that use concentrations of test compounds that are either too high (resulting in toxicity) or too low relative to the binding or dissociation constant of the compound to the target.
When a drug discovery scientist screens combinatorial mixtures of compounds, the scientist will conventionally identify an active pool, deconvolute it into its individual members, and identify the active members via re-synthesis and analysis of the discrete compounds. In addition to false positives and false negative, current techniques and protocols for the study of combinatorial libraries against a variety of biologically relevant targets have other shortcomings. These include the tedious nature, high cost, multi-step character, and low sensitivity of many screening technologies. These techniques do not always afford the most relevant structural and binding information, for example, the structure of a target in solution and the nature and the mode of the binding of the ligand with the receptor site. Further, they do not give relevant information as to whether a ligand is a competitive, noncompetitive, concurrent or a cooperative binder of the biological target""s binding site.
The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development that has the potential to accelerate the identification of lead compounds in drug discovery. Rapid and direct methods have been developed to identify lead compounds in drug discovery involving affinity selection and mass spectrometry. In this strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to develop a structural activity relationship (SAR) in order to optimize the binding characteristics of the ligand with the target receptor.
One step in the identification of bioactive compounds involves the determination of binding affinity and binding mode of test compounds for a desired biopolymeric or other receptor. For combinatorial chemistry, with its ability to synthesize, or isolate from natural sources, large numbers of compounds for in vitro biological screening, this challenge is greatly magnified. Since combinatorial chemistry generates large numbers of compounds, often isolated as mixtures, there is a need for methods which allow rapid determination of those members of the library or mixture that are most active, those which bind with the highest affinity, and the nature and the mode of the binding of a ligand to a receptor target.
An analysis of the nature and strength of the interaction between a ligand (agonist, antagonist, or inhibitor) and its target can be performed by ELISA (Kemeny and Challacombe, in ELISA and other Solid Phase Immunoassays: Theoretical and Practical Aspects; Wiley, New York, 1988), radioligand binding assays (Berson and Yalow, Clin. Chim. Acta, 1968, 22, 51-60; Chard, in xe2x80x9cAn Introduction to Radioimmunoassay and Related Techniques,xe2x80x9d Elsevier press, Amsterdam/New York, 1982), surface-plasmon resonance (Karlsson, Michaelsson and Mattson, J. Immunol. Methods, 1991, 145, 229; Jonsson et al., Biotechniques, 1991, 11, 620), or scintillation proximity assays (Udenfriend, Gerber and Nelson, Anal. Biochem., 1987, 161, 494-500). Radio-ligand binding assays are typically useful only when assessing the competitive binding of the unknown at the binding site for that of the radio-ligand and also require the use of radioactivity. The surface-plasmon resonance technique is more straightforward to use, but is also quite costly. Conventional biochemical assays of binding kinetics, and dissociation and association constants are also helpful in elucidating the nature of the target-ligand interactions but are limited to the analysis of a few discrete compounds.
A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science, 1996, 274, 5252, 1531). The approach is called SAR by NMR because structure-activity relationships (SAR) are obtained from NMR. This technique has several drawbacks for routine screening of a library of compounds. For example, the biological target is required to incorporate a 15N label. Typically the nitrogen atom of the label is part of amide moiety within the molecule. Because this technique requires deshielding between nuclei of proximal atoms, the 15N label must also be in close proximity to a biological target""s binding site to identify ligands that bind to that site. The binding of a ligand conveys only the approximate location of the ligands. It provides no information about the strength or mode of binding.
Therefore, methods for the screening and identification of complex target/ligand binding are greatly needed. In particular, new methods are needed for the identification of the strength and mode of binding of a ligand to its intended target.
This invention provides for methods and processes for identifying weak binding ligands for a target molecule. Ligands are selected that have an affinity for the target molecule that is equal to or greater than a baseline affinity. This can be accomplished according to one embodiment of the invention by utilizing a mass spectrometer and selecting a standard ligand that forms a non-covalent binding complex with the target molecule. An amount of the standard ligand is mixed with an excess amount of the target molecule such that unbound target molecule is present in the mixture. This mixture is introduced into the mass spectrometer and the operating performance conditions of the mass spectrometer are adjusted such that the signal strength of the standard ligand bound to the target molecule is from about 1% to about 30% of the signal strength of unbound target molecule. At least one further ligand is introduced into a test mixture of the target molecule and the standard ligand and this test mixture is introduced into the mass spectrometer. Any complexes of the further ligand and the target wherein the ligand has greater than baseline affinity for the target molecule is identified by discerning the signals that have a signal strength greater than the background noise of the mass spectrometer.
The invention further provides for methods and processes for selecting those members of a group of compounds that can form a non-covalent complex with a target molecule and where the affinity of the members for the target molecule is greater than a baseline affinity. This can be accomplished by utilizing a mass spectrometer and selecting a standard compound that forms a non-covalent binding complex with the target molecule. An amount of the standard compound is mixed with an excess amount of the target molecule such that unbound target molecule is present in the mixture and the mixture is introduced into the mass spectrometer. The operating performance conditions of the mass spectrometer are adjusted such that the signal strength of the standard compound bound to the target molecule is from about 1% to about 30% of signal strength of unbound target molecule. Next a sub-set of the group of compounds is introduced into a test mixture of the target molecule and standard compound and this test mixture is introduced into the mass spectrometer. Those members of the sub-set of compounds that form complexes with the target with an affinity greater than baseline are identified by discerning those signals that have a signal strength greater than the background noise of the mass spectrometer. The individual members are then identified by their respective molecular masses.
The invention further includes methods and processes for determining the relative interaction between at least two ligands with respect to a target substrate. This is accomplished by mixing an amount of each of the ligands with an amount of the target substrate to form a mixture. The mixture is then analyzed using mass spectrometry to determine the presence or absence of a ternary complex corresponding to simultaneous binding of two of the ligands with the target substrate. The absence of a ternary complex in the mixture indicates that binding of the ligands to the target is competitive while the presence of a ternary complex indicates that binding of the ligands is other than competitive.
The invention further includes methods and processes for determining the binding interaction of ligands to a target-substrate. This is accomplished by mass spectrometry analysis of the mixture as described to determine if the binding is other than competitive followed by determination of the ion abundance of i) a ternary complex present in the mixture, ii) a first binary complex corresponding,to the adduction of a first ligand with the target substrate; iii) a second binary complex corresponding to the adduction of a second ligand with the target substrate; and iv) target substrate unbound or not complexed with either of the first or second ligands. The absolute ion abundance of the ternary complex is compared to the sum of the relative ion abundance of the binary complexes which contribute to the formation of the ternary complex. The relative ion abundance of one of the contributing binary complexes is calculated by multiplying the absolute ion abundance of the first binary complex with the relative ion abundance of the second binary complex with respect to the unbound target substrate. The relative ion abundance of the second binary complex is calculated by dividing that binary complex"" absolute ion abundance by the absolute ion abundance of the unbound target. Similarly, the relative ion abundance of the other contributing binary complex is calculated by multiplying the absolute ion abundance of the second binary complex with the relative ion abundance of the first binary complex.
If the absolute ion abundance of the ternary complex is equal to the sum of the relative ion abundances of the contributing binary complexes this indicates concurrent binding interaction of the ligands to the target substrate. If the absolute ternary complex ion abundance is greater this indicates cooperative binding interaction, and if lesser this indicates competitive binding interaction.
The invention further includes methods and processes for determining the relative proximity of binding sites of a first and a second ligand on a target substrate. This can be accomplished by exposing the target substrate to a mixture of the second ligand and a plurality of derivative compounds of the first ligand. Each of the first ligand derivatives has the chemical structure of the first ligand and at least one substituent group pending from it or if the first ligand includes a ring within its structure, derivatives of the ligand can include expansion of contraction of that ring. This mixture is analyzed by mass spectrometry to identify a first ligand derivative that inhibits the binding of the second ligand to the target substrate or visa versa, i.e. binds competitively with the second ligand as determined by the absence of a ternary complex corresponding to the simultaneous complexation of the first ligand derivative and the second ligand with the target.
This invention further provides for methods and processes for determining the relative orientation of a first ligand to a second ligand when these ligands are bound to a target substrate. This is accomplished by exposing the target substrate to a mixture of the second ligand and a plurality of derivative compounds of the first ligand. Each of the first ligand derivatives has the chemical structure of the first ligand and a substituent group pending therefrom. The mixture is analyzed by mass spectrometry to identify a first ligand derivative that inhibits the binding of the second ligand to the target substrate or visa versa, i.e. binds competitively with the second ligand as determined by the absence of a ternary complex corresponding to the simultaneous complexation of the first ligand derivative and the second ligand with the target.
This invention further provides for a screening method for determining compounds that have binding affinity to a target substrate. This is accomplished using mass spectrometry to identifying two ligands that bind to a target non-competitively in a mixture of the ligands and target substrate. These two ligands are then concatenated to form another ligand that has greater binding affinity for the target substrate than either of the two ligands.
The invention further includes methods and processes for modulating the binding affinity of ligands for a target molecule. This is accomplished by selecting a first ligand fragment and a second ligand fragment and then exposing a target molecule to these ligand fragments. The target molecule exposed to the ligand fragments is then interrogated in a mass spectrometer to identify binding of the ligand fragments to the target molecules. The ligand fragments are concatenated together in a structural configuration that improves the binding properties of the fragments for the target molecule.
The invention further includes methods and processes for refining the binding of ligands to target molecules. This is accomplished by selecting first and second virtual fragments of a ligand followed by virtually concatenating the selected ligand fragments together in silico to form a 3D model of the concatenated ligand fragments. This 3D model of the concatenated ligand fragments is then positioned in silico on a 3D model of the target molecule. The various in silico positions of the 3D model of the concatenated ligand fragments on the in silico 3D model of the target molecule are scored. Using the results of the scoring, the in silico position of the 3D model of the concatenated ligand fragments on the in silico 3D model of the target molecule is refined. In a preferred embodiment of this method, real ligand fragments corresponding to the virtual ligand fragments are concatenated together to covalently join these ligand fragments into a new molecule. The new molecule is mixed with a target molecule and the mixture interrogated in the mass spectrometer for binding of the new molecule to the target molecule.
In each of the above methods and processes, in a preferred embodiment, an electrospray mass spectrometer is utilized. Preferred electrospray ionization is accomplished by Z-spray, microspray, off-axis spray or pneumatically assisted electrospray ionization. Further countercurrent drying gas can be used. Preferred mass analyzers for use in identifying the complexes are quadrupole, quadrupole ion trap, time-of-flight, FT-ICR and hybrid mass detectors. The preferred method of measuring signal strength is by the relative ion abundance. The mass spectrometer can also include a gated ion storage device for effecting thermolysis of the test mixtures within the mass spectrometer.
Adjustment of the mass spectrometer operating performance conditions would include adjustment of the source voltage potential across the desolvation capillary and a lens element of the mass spectrometer. This is best monitored by ion abundance of free target molecule. Adjustment of the mass spectrometer operating conditions further can include adjustment of the temperature of the desolvation capillary and adjustment of the operating gas pressure with the mass spectrometer downstream of the desolvation capillary.
In a preferred embodiment, adjustment of the operating performance conditions of the mass spectrometer is effected by adjustment of the voltage potential across the desolvation capillary and a lens element to generate an ion abundance of the ion from a complex of standard ligand with the target of from about 1% to about 30% compared to the abundance of the ion from the target molecule. A more preferred range of abundance of the complex of standard ligand with target to the abundance of the ion from the target molecule is from about 10% to about 20%.
Preferred for standard ligands are those ligands having a baseline affinity for the target of about 10 to about 100 millimolar. Particularly preferred are standard ligands having a baseline affinity for the target molecule of about 50 millimolar as expressed as a dissociation constant. Particularly preferred for standard ligands for nucleic acid targets are amines, primary, secondary or tertiary, amino acids, and nitrogen containing heterocycles with ammonium being the most preferred. Particularly preferred for standard ligands for peptides are esters, phosphates, borates, amino acid and nitrogen containing heterocycles.
The target molecule can be one of various target molecules including RNA, DNA, proteins, RNA-DNA duplexes, DNA duplexes, polysaccharides, phospholipids and glycolipids. Preferred are nucleic acids and proteins with RNA being particularly preferred as a target molecule.
Various RNA molecules are useful as the target. Preferred RNA target molecules are those that are fragments of larger RNA molecules including those being from about 10 to about 200 nucleotides in length. A more preferred RNA target is RNA of from about 15 to about 100 nucleotides in length including those having secondary and ternary structure.
Preferred ligand molecules include those having a molecular mass of less than about 1000 Daltons and fewer that 15 rotatable bonds, i.e., covalent bonds linking one atom to a further atom in the molecule and subject to rotation of the respective atoms about the axis of the bond. More preferred ligands molecules include those having a molecular mass of less than about 600 Daltons and fewer than 8 rotatable bonds. Even more preferred ligand molecules include those have a molecular mass of less than about 200 Daltons and fewer than 4 rotatable bonds. Further preferred ligands include those having no more than one sulfur, phosphorous or halogen atom.
The ligands can comprise members of collection libraries. Preferred collection libraries include historical repositories of compounds, collections of natural products, collections of drug substances or intermediates for such drug substances, collections of dyestuffs, commercial collections of compounds or combinatorial libraries of compounds. A preferred collection for selecting ligands can contain various numbers of members with libraries of from 2 to about 100,000 being preferred.